HeNe Laser (633 nm)-Coupled Confocal Microscope Allows Simulating Magnetic Resonance Imaging/Computed Tomography Scan of the Brain and Eye: A Noninvasive Optical Approach Applicable to Small Laboratory Animals

Introduction

Magnetic resonance imaging (MRI) and computed tomography (CT) are noninvasive medical imaging techniques used for the detailed visualization of internal organs, parts of organs, or tissues of the human body. The techniques are used for clinical purposes such as making a diagnosis or examining the severity or progress of particular diseases. Alternatively, these radiological applications are used for medical science, including the study of normal human anatomy and physiology. The noninvasive techniques of MRI or CT scanning are most commonly applied in clinical neurology and neurosurgery. Unfortunately, neither MRI nor CT is suitable to be applied as a noninvasive imaging tool for research purposes in small laboratory animals such as zebrafish embryos or larvae. Even the established micro-CT scanner is only suitable for scanning adult fishes,^1,2 and a staining procedure is required for imaging. ³ More importantly, micro-CT, just like medically used CT, offers a good image quality for bony tissues only and has much lower soft tissue contrast. ⁴ Therefore, when imaging soft tissues, the administration of an exogenous contrast agent is frequently desirable and sometimes indispensable. ⁴ Moreover, as CT uses X-rays for imaging, there is a considerable risk of radiation exposure with potential physical insult to human or animals. The introduction of laser techniques has enabled the study of spatial and temporal coherence of internal organs and tissues. Laser with its high spectral purity light sources has proven to be suitable for probing internal organs in humans or animals. ⁵ The laser-coupled confocal microscope is a powerful tool for detailing structures within a defined range of tissue depth. ⁵ The 633 nm wavelength HeNe laser is the best known and most widely used in the red part of the visible spectrum. The visible output of the red HeNe laser, long coherence length, and its excellent spatial quality makes this laser suitable to be used as a tool for holography of organs or tissues. In this study, we evaluated whether 633 nm HeNe laser can penetrate well into intact brain and eye of small animals such as zebrafish and whether the detection of differential signals from normal and pathological brain is possible with this noninvasive technique.

Materials and Methods

All zebrafish protocols were approved by the Institute of Animal Care.^6,7 Optical imaging using Zeiss LSM 510 confocal microscope via 633 nm HeNe laser line (Carl Zeiss) was used for imaging the intact brain and eyes of zebrafish embryos/larvae. Although the most important features of a confocal laser scanning microscope (CLSM) are its capability of isolating and collecting a plane of focus from within a fluorescent sample and precise colocalization for double- and triple-labeled samples, we did not apply the feature in this study. We used an alternative way by simply placing intact unstained zebrafish embryos/larvae into a plate under CLSM, followed by using 633 nm HeNe laser and optical sectioning properties for scanning. CLSM is an optical sectioning microscope. The imaging method requires a CLSM equipped with 633 nm HeNe laser. A microscope without optical sectioning properties and 633 nm HeNe laser is unsuitable for this method. The imaging method does not require any fluorescent or nonfluorescent dye stainings, and therefore, any staining procedure for detection is not required. To immobilize living zebrafish embryos or larvae, we fixed them in 4% paraformaldehyde as previously described ⁶ and arrayed them in a 24-well plate (one piece/well). To minimize the risk of drying out the samples during the scanning procedure, we used a plate cover throughout the scanning procedure. To differentiate normal brain from its pathological counterpart, we also implicated the technique to the brains and eyes of our zebrafish Glut1 knockdown model in which alterations of cerebral ⁷ and ocular ⁸ structures have taken place.

Results and Discussion

Generally, the optical properties of tissues/organs are determined by light scattering and the absorption coefficients of the particular area. ⁹ Changes in light scattering or absorption can provide useful information about the altered compositions and the optical properties of a particular region. We evaluated age-paired controls (n = 60) and Glut1 knockdown zebrafish embryos/larvae (n = 60) at various stages of postfertilization (days post-fertilization [dpf]). In this study, we focused on brain and eye of zebrafish embryos/larvae. The differential signals from normal and diseased zebrafish embryos/larvae are shown in Figure 1.

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FIG. 1.

Detection of differential signals of normal and diseased brain and eye of zebrafish. (A) A control brain/eye from a 2 days post-fertilization (dpf) zebrafish. (B) Structural defect of brain and eye is seen in a corresponding Glut1MOR. Some punched-out areas cover all brain regions. The eye is smaller than that of the control with defective retinal development. (C) A control brain/eye from a 3 dpf zebrafish. (D) A corresponding Glut1MOR shows similar structural defects of brain and eye as in photo B. The defects have caused a honeycomb pattern of the brain. (E) A control brain/eye from a 4 dpf zebrafish. (F) An increased diffuse light reflectance intensity (hyperintensity) of brain is noticed in a corresponding Glut1MOR when compared with the control in photo E. Regional hyperintensity (arrow) is noticed in the eye of the Glut1MOR in contrast to the healthy eye in photo E. FB, forebrain; MB, midbrain; HB, hindbrain; Glut1MOR, Glut1 morphant.

This study illustrates that the 633 nm HeNe laser penetrates deep enough to visualize an intact organ of a normal small laboratory animal. The detection of differential signals from the normal and pathological brain and eye is possible with this noninvasive technique. Using the 633 nm HeNe laser-coupled confocal microscope enables to adequately simulate imaging at least as good as the images of MRI/CT scanning of brain and eye. The optical method provided good spatial resolution, sufficient soft tissue contrast without using any contrast agents, noninvasive detection, and localization of soft tissue alterations in intact brain and eye of zebrafish embryos/larvae. We expect that the method may be further extended for the imaging of other organs/tissues of zebrafish or other small laboratory animals for research purpose.